As described in project 1, the highly conserved Wnt/beta-catenin signaling pathway is a crucial pathway for embryogenesis. Mutations in oncogenes or tumor suppressors in the Wnt/beta-catenin pathway lead to its constitutive activation and cause cancer in the adult. We have previously shown that alternative Wnt signaling pathways, controlled by noncanonical Wnts such as Wnt5a, also play crucial roles in embryo morphogenesis. In vitro studies separately reveal that Wnt5a can antagonize Wnt/beta-catenin signaling suggesting that stimulation of these alternative pathways could function to suppress tumorigenesis. Embryos lacking Wnt5a display defects in the elongation of the primary anterior-posterior body axis and presomitic mesoderm (PSM) that are distinct from the axis elongation defects observed in Wnt3a-/- phenotypes. Proper elongation of the PSM is important for correctly establishing the slope of the Wnt3a gradient that controls mesodermal stem cell homeostasis and somitogenesis. Although several pathways have been proposed to transduce Wnt5a signals, our genetic and in vivo reporter analyses demonstrate that Wnt5a interacts with core components of the Planar Cell Polarity (PCP) pathway (Vangl2 and Dvl2) to regulate PSM elongation. Many in vitro studies have suggested that Wnt5a negatively regulates the Wnt/betacatenin pathway however analyses of Wnt/betacatenin target genes and reporters in Wnt5a-/- embryos were not supportive. To address the mechanisms of Wnt5a signaling, we transcriptionally profiled Wnt5a mutants following the same procedure that we successfully used to profile Wnt3a mutants (see project 1). In contrast to our results with Wnt3a mutants, no consistent changes in gene expression were observed when comparing wt and Wnt5a-/- profiles suggesting that Wnt5a does not regulate transcription and does not inhibit the expression of Wnt3a/betacatenin target genes. Together, our data support the interpretation that Wnt3a and Wnt5a regulate distinct pathways in vivo. Furthermore, our data argues that Wnt5a signals, at least in part, through the PCP pathway, and that it does not antagonize the Wnt/betacatenin signaling pathway during early embryogenesis. We have recently published a paper examining the role of Wnt5a during organogenesis, specifically in the developing gastrointestinal tract (Cervantes et al., 2009). To better understand the function of Wnt5a in later development, and in normal and diseased adult stages, we have generated conditional loss and gain-of-function alleles of Wnt5a. Conditional knockouts of Wnt5a, specifically in posterior embryonic mesoderm and the heart outflow tract, recapitulate the kidney, gut and heart phenotypes and, as expected, rescues the facial defects. These results demonstrate that the heart phenotype arises strictly from a requirement for Wnt5a in the outflow tract, and not in other parts of the heart such as the myocardium. The kidney phenotypes are currently being analysed in collaboration with my colleague and kidney expert, Dr. Perantoni. Preliminary analyses of the Wnt5a gain-of-function mutant suggest that this allele is also working as expected, and interesting phenotypes have been observed when Wnt5a is overexpressed by tissue-specific Cre drivers. The conditional Wnt5a loss and gain of function alleles will be particularly useful in our attempts to address the role of this pathway in suppressing tumorigenesis. Experiments are underway to examine whether Wnt5a plays a role in gastrointestinal tumorigenesis. We are using the Villin-CreERT2 line to inducibly delete or overexpress Wnt5a in the GI tracts of Apcmin mice which develop hundreds of intestinal adenomas due to overactivation of the Wnt/beta-catenin pathway. If Wnt5a has a tumor suppressor role in GI tumors then we predict that tumor formation in Apcmin mice should be suppressed by Wnt5a overexpression, and exacerbated by a reduction in Wnt5a activity. In an effort to better understand the potential role of Wnt5a in PCP signaling, we attempted to develop an in vitro morphogenesis assay in ES cells by generating ES cells carrying epitope-tagged inducible Wnt5a, Vangl2, Ror2 (an atypical Wnt5a receptor), or Prickle. Several ES cell lines have been generated and are currently being characterized. Differentiating embryoid bodies generate self-organizing asymmetry and display polarized distribution of differentiated cell types. We will assess asymmetric gene expression, differentiated cell distribution, adhesion and embryoid body morphology upon overexpression of Wnt5a and PCP genes in embryoid bodies. Even if these cells fail to provide a convenient assay for morphogenesis, the epitope tags will prove to be useful for the identification of associated proteins by immunoprecipitation and mass spectroscopy analysis. We have also identified mouse homologues of Daam (Dishevelled (Dvl)-associated activator of morphogenesis), a Formin protein that promotes actin polymerization and which interacts directly with Dvl and Rho to control convergent extension in frogs. We have generated numerous tools including targeted conditional loss-of-function alleles of Daam1 and Daam2, LacZ-tagged alleles, epitope tagged cDNAs and antibodies to understand how the Daam proteins function in the Wnt pathway to regulate cell and tissue polarity during mouse embryogenesis. We have found that the loss of Daam1 function results in embryonic lethality at mid-gestation due to defects in the development of the labyrinth layer of the placenta. Although Daam2-/- animals are viable and fertile, Daam1-/-/Daam2-/- double mutants died earlier due to more severe placental phenotypes, suggesting that Daam1 and Daam2 have redundant functions in the placenta. Interestingly, loss of Daam1 function in Wnt5a null mutants leads to a neural tube closure defect (NTD) that is not observed in either single mutant. Since a NTD is characteristic of PCP mutant phenotypes, this result is consistent with Wnt5a and Daam1 participating in the PCP pathway. Our work suggests a novel function for the Wnt/PCP pathway in placental development. Since the placenta is a critical support structure for the developing fetus, our work could lead to ways to help prevent the complications arising from placental conditions such as premature birth and excessive maternal bleeding. We have written the first draft of a manuscript that we expect to submit shortly.